JP2021136936A - Cryopreservation jig - Google Patents

Abstract

To provide a cryopreservation jig which is excellent in preservative liquid absorbability and recovering property of cell or tissue and can easily perform adjustment operation of a volume of preservative liquid when performing freezing operation of cell or tissue.SOLUTION: A cryopreservation jig has, in either front or rear surface of a preservation liquid absorber, at least one kind of image patterns formed by discontinuous lines including: an image pattern consisting of a plurality of circles or ellipses with the same centroid and different diameter; an image pattern consisting of a plurality of polygons with the same centroid and different length between the centroid and apex; and an image pattern consisting of a diagram formed by combining a circle or an ellipse and a polygon with the same centroid.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cryopreservation jig for cryopreserving cells or tissues.

Excellent cell or tissue preservation techniques are sought after in various industrial fields. For example, in a bovine embryo transfer technique, embryos are cryopreserved, thawed and transplanted in accordance with the estrous cycle of a recipient cattle. Further, in human infertility treatment, after collecting an egg or an ovary from a mother, it is cryopreserved in order to adjust to a timing suitable for transplantation, and thawed at the time of transplantation for use.

In general, cells or tissues collected from a living body gradually lose their activity even in a culture solution, and thus long-term culture outside the living body is not preferable. Therefore, a technique for long-term storage without losing biological activity is important. Superior preservation techniques allow for more accurate analysis of harvested cells or tissues. In addition, excellent preservation technology makes it possible to use cells or tissues for transplantation while maintaining higher biological activity, and it is expected that the survival rate after transplantation will be improved. Furthermore, it is also possible to sequentially produce and store cultured skin cultured in vitro and artificial tissues for transplantation such as so-called cell sheets constructed in vitro and use them when necessary. Great benefits can be expected in both medical and industrial fields.

As a method for preserving cells or tissues, for example, a slow freezing method is known. In this method, cells or tissues are immersed in a preservation solution obtained by containing a freezing agent in a physiological solution such as, for example, phosphate buffered saline. As the antifreeze agent, compounds such as glycerol and ethylene glycol are used. After immersing the cells or tissues in the preservation solution, when the cells or tissues are cooled to -30 to -35 ° C. at a relatively slow cooling rate (for example, a rate of 0.3 to 0.5 ° C./min), the cells or tissues are inside or outside the cells or inside and outside the tissues. The storage solution is sufficiently cooled and becomes highly viscous. In such a state, when the cells or tissues in the preservation solution are further cooled with a cooling solvent (for example, liquid nitrogen), the minute solutions inside the cells or inside the tissues and around the outside become solids in an amorphous state. Vitrification occurs. When the inside and outside of cells or the inside and outside of tissues are solidified by vitrification, the movement of molecules is substantially stopped. Therefore, it is considered that the vitrified cells or tissues can be stored semi-permanently by storing them in liquid nitrogen.

For example, Patent Document 1 describes that porcine embryos are cryopreserved by a slow freezing method, and Patent Document 2 describes that bovine embryos are preserved by a slow freezing method.

However, the slow freezing method cools cells or tissues at a relatively slow cooling rate, so that the operation for cryopreservation takes time. In addition, there is a problem that a device or a jig for controlling the cooling rate is required. In addition, since ice crystals are formed in the extracellular or extracellular storage solution, the cells or tissues may be physically damaged by the ice crystals.

On the other hand, the vitrification freezing method is known as a storage method for solving the above-mentioned problems in the slow freezing method. The vitrification freezing method uses the principle that ice crystals are less likely to form even below freezing point due to the freezing point depression of a storage solution containing a large amount of freezing agent such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide). .. When this preservation solution is rapidly cooled in a cooling solvent, it can be solidified without forming ice crystals. This solidification is called vitrification freezing. A preservative solution containing a large amount of antifreeze used for vitrification freezing is called a vitrification solution.

In the vitrification freezing method, cells or tissues are immersed in a vitrifying solution and then rapidly cooled in a cooling solvent. Since the vitrification freezing method is such a simple and quick process, it does not require a long time for the operation for cryopreservation, and has the advantage that it does not require a device or jig for temperature control. There is.

Regarding cryopreservation of cells or tissues using the vitrification freezing method, examples using various types of cells or tissues are shown by various methods. For example, Patent Document 3 shows that the application of the vitrification freezing method to animal or human germ cells or somatic cells is extremely useful. Non-Patent Document 1 shows that the vitrification freezing method is effective for the preservation of Drosophila embryos. Further, Patent Document 4 shows that the vitrification freezing method is effective in preserving cultured plant cells and tissues.

However, the high concentration antifreeze contained in the vitrified solution is chemically toxic. Therefore, during cryopreservation of cells or tissues, it is desirable that the amount of vitrification solution existing around the cells or tissues is small, and it is desirable that the time for the cells to be exposed to the preservation solution, that is, the time until freezing is short. .. Furthermore, it is necessary to dilute the preservation solution immediately after thawing.

Patent Document 5 describes a cryopreservation jig having a strip-shaped flexible and colorless transparent film as an egg adhesion holding strip. In such a document, the amount of vitrified liquid around an egg or embryo is reduced by limiting the width of the egg attachment holding strip of the cryopreservation jig.

Patent Document 6 describes a cryopreservation jig including a base portion having light transmission as a living cell holding portion and a water absorbing portion fixed to the surface of the base portion. In such a document, the cryopreservation jig absorbs the excess vitrified liquid existing around the egg or embryo in the water absorbing part, which exceeds the range of the base part, thereby increasing the amount of the vitrified liquid. Is being reduced.

Patent Document 7 describes a cryopreservation jig having a preservative liquid removing material having through holes in a film-like material made of synthetic resin and a natural product such as wire mesh or paper. In such a document, excess vitrified liquid adhering to the periphery of an egg or embryo is removed by suction from the lower part of a preservative liquid removing material contained in the cryopreservation jig.

Patent Document 8 provides a cryopreservation jig as a jig for cryopreservation, which can remove an excess preservative solution containing a vitrification solution from the lower part without sucking, and has improved workability when placing cells or tissues. A jig for cryopreservation using a storage liquid absorber having a specific haze value is described. Further, Patent Document 9 describes a cryopreservation jig having a porous structure having a specific refractive index as a preservation solution absorber, and Patent Document 10 describes a preservation solution absorption containing fibers having a specific diameter. A jig for cryopreservation having a body is described.

Further, as a cryopreservation jig that facilitates the recovery of freeze-thawed cells or tissues, Patent Document 11 describes a placement portion position area and cells on which the cells or tissues are placed on the placement portion of the cells or tissues. Alternatively, a cryopreservation jig having a marking portion indicating the boundary of the non-placement area on which the tissue is not placed is described.

Japanese Unexamined Patent Publication No. 9-122158 Japanese Unexamined Patent Publication No. 2005-261413 Japanese Patent No. 3044323 Japanese Unexamined Patent Publication No. 2008-5846 Japanese Unexamined Patent Publication No. 2006-271395 International Publication No. 2019/004300 Pamphlet International Publication No. 2011/070973 Pamphlet Japanese Unexamined Patent Publication No. 2014-183757 International Publication No. 2015/064380 Pamphlet JP 2015-188405 Registered Utility Model No. 3202359

Steponkus et al. , Nature 345: 170-172 (1990)

The cryopreservation jig described in Patent Document 5 has a problem that it is difficult to place an egg or an embryo on a film having a limited width together with a very small amount of a vitrifying solution. Further, in the cryotop (registered trademark) method based on the method, the egg or embryo was once placed on the film together with the vitrification solution in order to cryopreserve the egg or embryo with a smaller amount of the vitrification solution. Later, a more complicated operation such as sucking the excess vitrifying liquid and removing it from the film may be performed.

In the cryopreservation jig described in Patent Document 6, the excess vitrified liquid around the egg or embryo on the base portion is not always sufficiently absorbed by the water absorbing portion.

In the cryopreservation jig described in Patent Document 7, a suction operation from the lower part is required when removing the excess vitrification liquid, and this operation is complicated.

Although all of the above-mentioned problems have been solved in the cryopreservation jigs described in Patent Documents 8 to 10, when cells or tissues are placed on the preservation solution absorber together with the preservation solution, the cells or tissues In some cases, the recoverability of the embryo was lowered because the amount of the preservative solution in the vicinity of the cell was excessively absorbed and became too small.

In the cryopreservation jig described in Patent Document 11, the recoverability of embryos is further improved by marking, but it is difficult to adjust the amount of preservative solution around cells or tissues to an appropriate amount, and further. There was a need for improvement.

An object of the present invention is to provide a cryopreservation jig which is excellent in storage liquid absorbability and cell or tissue recovery, and can easily perform a storage liquid amount adjustment operation in a cell or tissue freezing operation. And.

As a result of diligent studies to solve the above problems, the present inventors have found that the above problems can be solved by a cryopreservation jig having the following configuration.

The placement portion on which the cells or tissues are placed has a porous preservation solution absorber, and is composed of a plurality of circles or ellipses having the same center of gravity and different diameters on either the front or back surface of the preservation solution absorber. Image pattern, image pattern composed of multiple polygons with the same center of gravity but different lengths of the center of gravity and vertices, or image pattern composed of a combination of circles or ellipses and polygons with the same center of gravity. A cryopreservation jig having at least one of the above, wherein the image pattern is formed by discontinuous lines.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a cryopreservation jig which is excellent in preservative liquid absorption and cell or tissue retrievability, and can easily perform a preservative liquid amount adjustment operation in a cell or tissue freezing operation. can.

It is an overall view which shows an example of the cryopreservation jig of this invention. It is an enlarged view around the mounting part of the cryopreservation jig of FIG. It is an enlarged view around the mounting part which showed another example of the cryopreservation jig of this invention. It is an enlarged view around the mounting part which showed another example of the cryopreservation jig of this invention. It is an enlarged view around the mounting part which showed another example of the cryopreservation jig of this invention. It is an enlarged view around the mounting part which showed another example of the cryopreservation jig of this invention. It is an enlarged view around the mounting part of the cryopreservation jig produced in Comparative Example 1. It is an enlarged view around the mounting part of the cryopreservation jig produced in Comparative Example 2.

The cryopreservation jig of the present invention will be described in detail below.

The cryopreservation jig of the present invention is used for cryopreserving cells or tissues. Examples of such a cryopreservation protocol include the slow freezing method described above and the vitrification freezing method.

In the present invention, a cell includes not only a single cell but also a cell population composed of a plurality of cells. The cell population composed of a plurality of cells may be a cell population composed of a single type of cell or a cell population composed of a plurality of types of cells. The tissue may be a tissue composed of a single type of cells or a tissue composed of a plurality of types of cells, and may contain a non-cellular substance such as an extracellular matrix in addition to the cells. But it's okay.

Examples of cells that can be cryopreserved using the cryopreservation jig of the present invention include eggs, embryos, and sperms of mammals (for example, humans, cows, pigs, horses, rabbits, rats, mice, etc.). Germ cells such as; pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). Examples thereof include cultured cells such as primary cultured cells, subcultured cells, and cell line cells. The cells, in one or more embodiments, are fibroblasts, cancer-derived cells such as pancreatic and hepatic cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, and immunity. Adhesive cells such as cells can be mentioned. Furthermore, tissues that can be cryopreserved include tissues composed of allogeneic or heterologous cells, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium.

The jig for cryopreservation of the present invention is not limited to tissues directly collected from a living body, for example, cultured skin cultured and grown in vitro, a so-called cell sheet constructed in vitro, and Japanese Patent Application Laid-Open No. 2012-205516. It can also be suitably used for cryopreservation of artificial tissues such as the proposed tissue model having a three-dimensional structure.

The cryopreservation jig of the present invention has a porous storage liquid absorber as a placement portion on which cells or tissues are placed. "Porosity" in the present invention means a structure having pores (pores), and the preservation solution absorber may have continuous pores on the surface and inside of the preservation solution absorber. preferable. Examples of the storage liquid absorber include absorbent materials such as a fiber sheet, a porous resin sheet, a porous metal sheet, and a porous metal oxide sheet. From the viewpoint of ease of handling, the mounting portion is preferably in the shape of a strip or a sheet.

In the present invention, a non-woven fabric is exemplified as a sheet made of fibers used as a storage liquid absorber. The fibers contained in the non-woven fabric include cellulose fibers, rayon fibers and cupra fibers which are regenerated fibers made of cellulose fibers, acetate fibers which are semi-synthetic fibers from cellulose fibers, polyester fibers, nylon fibers, acrylic fibers and polypropylene. Examples thereof include fibers, polyethylene fibers, polyvinyl chloride fibers, vinylidene fibers, polyurethane fibers, vinylon (polyvinyl alcohol) fibers, glass fibers, silk fibers and the like, and non-woven fabrics obtained by mixing various of these fibers can also be used. Of these, rayon fibers and cupra fibers, which are cellulose fibers and fibers derived from cellulose fibers and are regenerated cellulose fibers, and acetate fibers, which are semi-synthetic fibers from cellulose fibers, are preferable.

The non-woven fabric described above preferably has a density of 0.1 to 0.4 g / cm ³ and a basis weight of 10 to 130 g / m ² . In particular, a non-woven fabric having a density of 0.12 ^{to 0.3 g / cm 3} and a basis weight of 10 to 100 g / m ² is excellent in absorption of a storage solution and further excellent in visibility of cells or tissues. It is preferable because it is possible to provide a storage jig.

The non-woven fabric preferably has a binder component such as a binder in an amount of 10% by mass or less, more preferably 5% by mass or less, and further preferably 3% by mass or less. In particular, a non-woven fabric containing no binder is preferable.

Unlike paper, there are various manufacturing methods for non-woven fabrics, but as the non-woven fabrics with reduced binder components described above, non-woven fabrics manufactured by the spunbond method and the melt blow method, and further, fibers are arranged by a wet method or a dry method. After that, a non-woven fabric produced by the water flow confounding method or the needle punching method can be preferably used. Further, as described above, the preferable fibers contained in the non-woven fabric in the present invention are cellulose fibers, fibers derived from cellulose fibers such as rayon fibers and cupra fibers which are regenerated cellulose fibers, and acetate fibers which are semi-synthetic fibers from cellulose fibers. However, in the case of manufacturing using these fibers, the manufacturing method by the water flow entanglement method or the needle punching method is preferable regardless of the wet method or the dry method.

The porous resin sheet used as the preservation solution absorber in the present invention is described in, for example, Japanese Patent Application Laid-Open No. 42-13560 and Japanese Patent Application Laid-Open No. 08-283447, which is stretched at least in the uniaxial direction to exceed the melting point of the resin. A resin sheet having a porous structure formed by a fine fibrous structure obtained by heating and sintering, and a thermoplastic resin obtained by a method such as emulsion polymerization or pulverization described in JP-A-2009-235417. Examples thereof include a resin sheet having a porous structure formed by filling a mold with solid powder, heating and sintering, and fusing and cooling the surface of powder particles. When the porous resin sheet is used as a preservation solution absorber, it is preferable because it is possible to provide a cryopreservation jig having excellent visibility of cells or tissues in addition to good absorption of the preservation solution. ..

Examples of the resin forming the above-mentioned porous resin sheet include various polyethylenes such as low-density polyethylene, high-density polyethylene, and ultra-high molecular weight polyethylene, and fluorine such as polypropylene, polymethylmethacrylate, polystyrene, polytetrafluoroethylene, and polyvinyldifluorolide. Examples thereof include resins, ethylene-vinyl acetate copolymers, polyamides, styrene-acrylonitrile copolymers, styrene-butadiene-acrylonitrile ternary copolymers, polycarbonates and polyvinyl chlorides. Among them, the porous resin sheet of fluororesin such as polytetrafluoroethylene or polyvinylidene difluoride allows the cells or tissues to be visually recognized under transmission microscope observation when the cells or tissues are placed on the resin sheet together with the preservation solution. The properties are dramatically improved, and it becomes possible to provide a cryopreservation jig having particularly excellent visibility of cells or tissues. Further, as the porous resin sheet, a membrane filter for filtration, which is commercially available for physics and chemistry experiment use and research use, can also be used.

Examples of the porous metal sheet include a porous metal sheet made of a metal such as copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, tin, zinc, lead, titanium, nickel and stainless steel. .. As the porous metal oxide sheet, a porous metal oxide sheet made of an oxide of a metal such as silica, alumina, or zirconium can be preferably used. Further, the porous metal sheet and the porous metal oxide sheet may be a porous sheet containing two or more kinds of the above-mentioned metal and metal oxide.

As a method for producing the above-mentioned porous metal sheet and the porous metal oxide sheet, a generally known method can be used. In the case of a porous metal sheet, a method such as a powder metallurgy method or a spacer method can be used. Further, the so-called powder space holder method, which is a combination of resin injection molding and powder metallurgy, can also be preferably used. For example, the pamphlet of International Publication No. 2006/041118, the method described in Japanese Patent No. 4578062, and the like can be used. More specifically, the metal powder and the resin to be the spacer are mixed, molded under pressure, and then fired in a high temperature environment to bake the metal powder, vaporize the resin to be the spacer, and make it porous. A sex metal sheet can be obtained. When the powder space holder method or the like is used, a resin binder can be mixed in addition to the metal powder and the resin serving as a spacer. Further, a method for producing a metal porous body such as a foam melting method or a gas expansion method in which a metal powder is heated at a high temperature and then gas is injected to create voids can also be used. Furthermore, a production method such as a slurry foaming method for producing a metal porous body using a foaming agent can also be used. When the storage liquid absorber is a porous metal oxide sheet, for example, the methods described in JP-A-2009-29692 and JP-A-2002-160930 can be used.

In the present invention, the pore size of the storage solution absorber is preferably 0.02 to 20 μm, more preferably 0.02 to 5 μm. If the pore size is less than 0.02 μm, the absorption performance of the storage solution may not be sufficient. In addition, there is a problem that it is difficult to manufacture a porous sheet. On the other hand, if the pore size exceeds 20 μm, the visibility of cells or tissues may be reduced. The pore diameter of the porous body in the present invention is the diameter of the largest pore measured by the bubble point test.

In the present invention, the porosity of the storage solution absorber is preferably 20% by volume or more, more preferably 30% by volume or more. It is preferable that the pores inside the storage liquid absorber have a continuous structure not only in the thickness direction but also in the direction perpendicular to the thickness direction. With such a structure, the pores inside the preservation solution absorber can be effectively used, so that high absorption performance of the preservation solution can be obtained. The thickness and porosity of the storage solution absorber can be appropriately selected depending on the type of cells or tissues used, the amount of the preservation solution dropped together with the cells or tissues, and the like.

The above-mentioned porosity is defined by the following formula. Here, the void volume V is the cumulative pore volume (ml / g) from a pore radius of 3 nm to 400 nm in the preservative solution absorber, which was measured and processed using a mercury porosimeter (measuring instrument name Autopore II 9220 manufacturer micromerits instrument corporation). ) Is multiplied by the dry solid content (g / square meter) of the preservative solution absorber, and can be obtained as a numerical value per unit area (square meter).
P = (V / T) x 100 (%)
P: Porosity (%)
V: Void volume (ml / m ² )
T: Thickness (μm)

The area of the preservation solution absorber may be appropriately set according to the amount of the preservation solution dropped together with the cells or tissues, and is not particularly limited. For example, ^{it may be 1 mm 2} or more per 1 μl of the preservation solution to be dropped. It is preferably ^{2 to 400 mm 2,} and more preferably 2 to 400 mm 2.

In the cryopreservation jig of the present invention, the mounting portion can be composed of only the storage liquid absorber, but can also be combined with a support that supports the storage liquid absorber. Examples of the support include various resin sheets, metal sheets, flat glass sheets, rubber sheets and the like. When the storage liquid absorber is a material such as a porous resin sheet whose light transmittance improves as the storage liquid is absorbed, if the support has a light transmittance of 80% or more in total light transmittance, the mounting portion Increased visibility of the cells or tissues above is particularly preferred. Examples of the light-transmitting support include various resin films, glass, rubber, and the like, and two or more kinds of light-transmitting supports may be used in combination as long as the effects of the present invention are not impaired. Among the above, the resin film is easy to handle and is preferably used. Specific examples of the resin film include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, polycarbonate resins, polymethylmethacrylate, methylmethacrylate-styrene copolymers, acrylic resins such as methylmethacrylate-polycarbonate copolymers, and low densities. Various polyethylenes such as polyethylene, high-density polyethylene and ultra-high molecular weight polyethylene, polypropylene, polystyrene, fluororesins such as polytetrafluoroethylene and polyvinyl difluororide, polyvinyl chloride, epoxy resin, silicone resin, diacetate resin, triacetate resin, Examples thereof include polyarylate resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, and polyolefin resin. The above-mentioned total light transmittance can be measured according to JIS-K-7361-1: 1997, for example, by the above-mentioned haze meter HZ-V3 of Suga Test Instruments Co., Ltd.

It is preferable to provide an adhesive layer between the above-mentioned storage liquid absorber and the support. The adhesive layer preferably contains a water-soluble polymer compound or a water-insoluble resin, which will be described later, or an instant adhesive composition, a hot melt adhesive composition, or a photocurable adhesive such as a moisture-curable adhesive substance. It is also possible to contain a composition or the like. For example, examples of the water-soluble polymer compound include polyvinyl alcohol, hydroxycellulose, polyvinylpyrrolidone, and starch paste, and examples of the water-insoluble resin include vinyl acetate resin, acrylic resin, epoxy resin, and urethane resin. Elastomer-based resin, cyanoacrylate-based resin, fluorine-based resin, silicon-based resin, nitrocellulose-based resin, nitrile rubber-based resin, styrene-butadiene-based resin, urea-based resin, styrene-based resin, phenol-based resin, polyimide-based resin, polyamide Examples thereof include based resins, polyester resins, bismaleimide resins, olefin resins, EVA resins and the like. The adhesive layer may contain one kind of resin or may contain a plurality of kinds of resins. Solid content of the adhesive layer is preferably in the range of 0.01 to 100 g / ^{m 2,} further range of 0.1 to 50 g / ^{m 2} is more preferable.

It is preferable that the above-mentioned adhesive layer is provided in the peripheral portion of the image pattern of the present invention having the mounting portion described later. If the adhesive layer is present in the vicinity of the image pattern, it may adversely affect the visibility of cells or tissues under microscopic observation, or may reduce the storage solution absorbability of the storage solution absorber. As a specific example for providing an adhesive layer around the image pattern of the mounting portion, masking is performed at a position on the support where the storage liquid absorber is provided and where the image pattern is further arranged. Masking with tape is applied, an adhesive layer is applied, and then the masking tape is peeled off to provide an adhesive layer at a position that becomes a peripheral portion of the image pattern, and a storage liquid absorber is attached onto the adhesive layer. The method is exemplified.

In the cryopreservation jig of the present invention, an image pattern composed of a plurality of circles or ellipses having the same center of gravity but different diameters and the same center of gravity are provided on either the front or back surface of the storage liquid absorber of the mounting portion described above. It has at least one kind of an image pattern composed of a plurality of polygons having different lengths of the center of gravity and the apex, or an image pattern composed of a figure formed by combining a circle or an ellipse having the same center of gravity and a polygon. Here, the front surface of the preservation solution absorber on either the front or back surface is the surface on which the cells or tissues are placed together with the preservation solution, and the back surface is the surface on the opposite side on which the cells or tissues are not placed. If the storage liquid absorber has the above-mentioned support, the back surface corresponds to the surface on the side to be bonded to the support. The center of gravity in the present invention is defined as a flat plate having a constant mass distribution with the sides of the circles, ellipses, or polygons having the above-mentioned image patterns as the outer circumferences, and is supported at that point. Means a point on a flat surface that can be balanced horizontally.

Examples of image patterns consisting of multiple circles or ellipses with the same center of gravity but different diameters include image patterns such as double circles, triple circles, and quadruple circles, and the same size ratio between major axis and minor axis. An example is an image pattern in which a plurality of ellipses having different diameters are combined, or an image pattern in which a plurality of ellipses having different ratios of major axis to minor axis are combined. When the preservation solution absorber has the above-mentioned image pattern, when the cells or tissues are placed on the placement portion together with the preservation solution, the operator gradually observes how the preservation solution is absorbed by the preservation solution absorber. It becomes possible to easily adjust the amount of the storage liquid at the time of freezing. The adjustment of the amount of the preservation solution is specifically the preservation existing around the cells or tissues in the process of being absorbed by the preservation solution absorber in the process of absorbing the preservation solution existing around the cells or tissues on the placement part. It refers to measuring the timing when the amount of liquid becomes appropriate, and the placement part is placed in a refrigerant such as liquid nitrogen at the timing when the storage liquid is not excessively absorbed by the storage liquid absorber and exists in an appropriate amount around cells or tissues. Immersion improves the recoverability of cells or tissues. During the operation, the preservative solution spreads in a circle on the preservative solution absorber and is absorbed. Therefore, it is most preferable in terms of operation that the image pattern is composed of a plurality of circles having the same center of gravity and different diameters. When the image pattern is composed of ellipses, the ratio of the major axis to the minor axis is preferably 3: 1 or less, and more preferably 2: 1 or less.

The number of vertices of individual polygons of an image pattern composed of a plurality of polygons having the same center of gravity but different lengths of the center of gravity and vertices may be the same or different. The polygonal shape of the image pattern includes regular polygons such as regular triangles, squares, regular pentagons, regular hexagons, regular hexagons, and regular octagons, triangles such as isosceles triangles and right angle triangles, and rectangles and parallel quadrilaterals. A polygon such as a quadrangle, a pentagon that is not a regular polygon, a hexagon, a hexagon, or an octagon is exemplified. Among them, it is preferable that a plurality of polygons are similar because it is easy to gradually recognize how the preservation solution is absorbed by the preservation solution absorber, and further, the plurality of polygons are similar. , When the polygon is a regular polygon having an even number of vertices, such as a square, a regular hexagon, and a regular octagon, it is particularly preferable in terms of operation. When the image pattern is composed of polygons other than regular polygons, the ratio of the maximum length to the minimum length between the center of gravity and each vertex is preferably 3: 1 or less, and more preferably 2: 1 or less.

As an example of an image pattern composed of a circle having the same center of gravity or a figure obtained by combining an ellipse and a polygon, an image pattern (for example, a square) in which the above-mentioned circle and the above-mentioned polygon are overlapped so as to have the same center of gravity. An image pattern in which a circle having a diameter smaller than the length of one side of the square is superimposed so that the center of gravity is the same), or the above-mentioned ellipse and the above-mentioned polygon are superimposed so that the center of gravity is the same. An example can be exemplified of a combined image pattern (for example, an image pattern in which a square having a side length smaller than the minor axis of the ellipse is superimposed on the ellipse so that the center of gravity is the same).

When the image pattern is a single pattern as described in Patent Document 11, it is difficult to adjust the amount of the storage solution.

The size of the image pattern and the number of patterns to be arranged may be appropriately set according to the size of the cells or tissues to be placed, but when the cells or tissues to be placed are small and the area of the storage solution absorber is also small. If the number of the image patterns arranged is too large, it may be difficult to describe the image patterns. For example, when the purpose is to place a mouse embryo (diameter of about 100 μm), the area of the preservation solution absorber is about 1.5 mm × 25 mm, and the largest outer circle is placed on the preservation solution absorber. If a double or triple circle image pattern with a diameter of about 1 mm and the smallest inner circle with a diameter of about 0.3 mm is provided, the operator can arbitrarily adjust the amount of storage liquid to be placed together with the cells or tissues. It is more preferable because it can be performed at a fine level and the image pattern can be easily depicted.

In the present invention, the individual circles, ellipses, and polygons of the image pattern described above are formed by discontinuous lines. Examples of discontinuous lines in the present invention include lines such as chain lines, broken lines, and dotted lines. Here, the chain line is a line in which short straight lines and points alternate, and a one-dot chain line and a two-dot chain line are exemplified. The broken line is a line provided with gaps at regular intervals. The dotted line is a line provided by displaying dots at regular intervals. If the discontinuous line is a broken line or a dotted line, the preservation solution absorber can absorb the preservation solution uniformly (and radially), and the amount of the preservation solution at the time of freezing can be easily adjusted. ,preferable. Further, the total length of the non-image portion of the discontinuous line (for example, the blank portion between the dots of the dotted line) is 25% or more of the total length of the image pattern, which means that the absorption of the preservation solution is high. Preferred from the point of view. If the image pattern is not formed by discontinuous lines, good storage solution absorbability will not be obtained. The line width of the image pattern is preferably 10 to 250 μm.

As a processing method for forming the above-mentioned image pattern on the storage liquid absorber, a method of pressing a mold having a convex pattern on either the front or back surface of the storage liquid absorber, or a method of applying the image pattern to a pigment. Alternatively, a plurality of methods depending on the material of the storage liquid absorber, such as a method of printing fine lines with dye ink, are exemplified, but from the viewpoint of not reducing the absorbability of the storage liquid absorber, the storage liquid absorber is convex. A method of pressing a mold having a pattern is preferable. Further, in the case of the method of pressing the mold having a convex pattern, the preservation solution is prevented from forming a convex portion on the surface of the preservation solution absorber (the surface on which the cells or tissues are placed together with the preservation solution). It is preferable to process from the surface of the absorber. If there is a protrusion on the surface of the preservation solution absorber, operability (for example, operability when cells or tissues are dropped and adhered to the preservation solution absorber together with the preservation solution at the time of freezing, or cells from the preservation solution absorber at the time of thawing Or, the operability when collecting the tissue) may be reduced. On the other hand, when the image pattern is formed by fine line printing using the pigment or dye ink described above, the back surface of the storage solution absorber (cell or tissue) is used to avoid the influence of the pigment or dye ink on the cells or tissues. It is preferable to process the non-mounting surface).

The cryopreservation jig of the present invention may have a grip portion together with the mounting portion described above. Having a grip portion is preferable because it improves workability during cryopreservation work and thawing work. The grip portion is preferably made of a liquid nitrogen resistant material. As such a material, for example, various metals such as aluminum, iron, copper, and stainless alloy, ABS resin, polypropylene resin, polyethylene resin, fluororesin, various engineering plastics, and glass can be preferably used.

When cells or tissues are cryopreserved for a long period of time using the cryopreservation jig of the present invention, the cryopreservation jig is covered with a cap in order to block the cells or tissues from the outside world, or the cryopreservation jig is used. It can be placed in a container of any shape and sealed. If liquid nitrogen is not sterilized and cells or tissues are brought into direct contact with liquid nitrogen to freeze, the sterilized state may not be guaranteed even if the cryopreservation jig is sterilized. Therefore, by capping the storage solution absorber to which the cells or tissues are attached before freezing, or by sealing the cryopreservation jig in the container, the cells or tissues are frozen without being in direct contact with liquid nitrogen. Sometimes. For the above reasons, it is preferable that the cap and the container are made of various metals, various resins, glass, ceramics, etc., which are materials having liquid nitrogen resistance. The shape is not particularly limited, and for example, the cap is brought into contact with a storage liquid absorber of a cryopreservation jig, such as a semi-spindle-shaped or dome-shaped cap such as a pencil cap, or a columnar straw cap. However, any shape may be used as long as it can block cells or tissues from the outside world. The shape of the container is not particularly limited as long as it can be sealed by encapsulating or storing the cryopreservation jig without contacting the placed cells or tissues.

The cryopreservation jig of the present invention can be used in combination with the above-mentioned cap and container as long as the effect of the present invention is not impaired. In addition, such a cryopreservation jig in a form used in combination with a cap or a container is also included in the present invention.

The cryopreservation jig of the present invention is preferably used in, for example, the cryotop method. Also, while conventional cryotop methods are typically used to conserve a single cell or a small number of cells less than 10, the present invention conserves more cells (eg, of 10 to 10,000,000 cells). It can also be suitably used in storage).

The procedure for cryopreserving cells or tissues using the cryopreservation jig of the present invention is not particularly limited. An example is shown below. First, the cells or tissues immersed in the preservation solution are dropped onto the placement portion (preservation solution absorber) together with the preservation solution. Although excess preservative solution is attached around the cell or tissue, the cell or tissue can be treated while checking the amount of preservative solution around the cell or tissue by using the image pattern of the present invention described above. The cells or tissues are frozen by immersing them in a refrigerant such as liquid nitrogen at an appropriate timing while holding them in the placement portion. At this time, a cap capable of blocking cells or tissues on the placement portion from the outside world may be attached to the storage solution absorber, or a cryopreservation jig may be sealed in the container. As the storage solution, those usually used for freezing cells such as eggs and embryos can be used. For example, a freezing agent (glycerol, ethylene glycol, etc.) is used in a physiological solution such as phosphate buffered saline. A large amount of various antifreeze agents such as glycerol, ethylene glycol, and DMSO (dimethylsulfoxide) (at least 10% by mass or more, more preferably 20% by mass, based on the total mass of the preservation solution). % Or more) can be used as a preservative solution. In the thawing operation, the cryopreservation jig of the present invention is taken out from a cooling solvent such as liquid nitrogen, and the mounting portion on which the frozen cells or tissues are placed is immersed in the thawing solution, if necessary. The cell or tissue is collected by shaking the preservative solution absorber in the melt.

Hereinafter, the cryopreservation jig of the present invention will be described in more detail with reference to the drawings.

FIG. 1 is an overall view showing an example of the cryopreservation jig of the present invention. In FIG. 1, the cryopreservation jig 3 has a mounting portion 2 connected to a grip portion 1.

A method of connecting the grip portion 1 and the mounting portion 2 of FIG. 1 will be described. When the grip portion 1 is made of resin, for example, a self-supporting mounting portion (a mounting portion having no support and consisting only of a storage liquid absorber) 2 is connected by insert molding at the time of molding the grip portion 1. can do. Further, a structure insertion portion (not shown) can be formed in the grip portion 1 and the mounting portion 2 can be connected with an adhesive. Although various adhesives can be used, silicon-based or fluorine-based adhesives that are resistant to low temperatures can be preferably used.

FIG. 2 is an enlarged view of the periphery of the mounting portion of the cryopreservation jig of FIG. In FIG. 2, the mounting portion 2 has an image pattern 4a composed of three circles (circles formed by dotted lines) having the same center of gravity and different diameters.

FIG. 3 is an enlarged view of the periphery of the mounting portion showing another example of the cryopreservation jig of the present invention. In FIG. 3, the mounting portion 2 has an image pattern 4b composed of two squares (squares formed by dotted lines) having the same center of gravity but different lengths of the center of gravity and the apex.

FIG. 4 is an enlarged view of the periphery of the mounting portion showing another example of the mounting portion in the cryopreservation jig of the present invention. The mounting portion 2 has an image pattern 4c composed of a regular hexagon having the same center of gravity (a regular hexagon formed by a dotted line) and a square (a square formed by a dotted line).

FIG. 5 is an enlarged view of the periphery of the mounting portion showing another example of the mounting portion in the cryopreservation jig of the present invention. The mounting portion 2 is composed of a square (a square formed by a dotted line) and a circle (a circle formed by a dotted line) having the same center of gravity, and is composed of a circle having a diameter smaller than the length of one side of the square. It has an image pattern 4d.

FIG. 6 is an enlarged view of the periphery of the mounting portion showing another example of the cryopreservation jig of the present invention. The mounting portion 2 has an image pattern 4e composed of two ellipses (two ellipses formed by dotted lines) having the same center of gravity, different major diameters and minor diameters, and having a similar relationship.

Hereinafter, the present invention will be described in more detail with reference to Examples, and the present invention will be specifically described, but the present invention is not limited to the following Examples.

(Example 1)
As a support to support the storage solution absorber, Toray Industries, Inc.'s polyethylene terephthalate film, Lumirror (registered trademark) T60 (thickness 188 μm, total light transmittance 89%), and masking tape manufactured by Teraoka Seisakusho Co., Ltd. Masking was performed according to (thickness 55 μm). The masking position was set to the position where the image pattern was formed on the storage solution absorber in the subsequent step, and the masking range was set to the range of the size in which all the image patterns were included. The masked polyethylene terephthalate film was coated with Henkel Japan Ltd. hot melt urethane resin Purmelt (registered trademark) QR 170-7141P as an adhesive layer so that the solid content at the time of drying was 30 g / m ² . .. After applying the adhesive layer, the masking tape is peeled off, and before the adhesive layer is cured, a polytetrafluoroethylene porous body manufactured by Advantech Toyo Co., Ltd. (pore diameter 0.2 μm, porosity 71%, thickness 35 μm) is used as a preservative solution absorber. ) Was pasted together. The obtained preservative solution absorber and the bonded body of the support were aged at room temperature for 24 hours to promote the curing of the adhesive layer. Then, the bonded body was cut into a rectangle having a short side of 1.5 mm and a long side of 25 mm. At this time, the rectangle was cut so as to include a position having no adhesive layer closer to one end (short side) than the center in the long side direction. The cut bonded body is fixed, and the image pattern 4a (three circles having the same center of gravity but different diameters (diameter 50 μm) shown in FIG. A circle)) formed by a dotted line composed of dots was formed by pressing the mold. The diameter of the circle of the image pattern was 0.75 mm for the outermost circle, 0.5 mm for the second outer circle, and 0.25 mm for the innermost circle. The total length of the non-image area of the pattern was set to 50% of the total length of the image pattern. The jig for cryopreservation of Example 1 was obtained by joining the end portion (short side portion) side of the bonded body without the image pattern to the grip portion made of ABS resin.

(Example 2)
In the production of the cryopreservation jig of Example 1, the image pattern 4b (two squares having the same center of gravity but different lengths between the center of gravity and each vertex (squares formed by dotted lines)) shown in FIG. 3 is formed. A jig for cryopreservation of Example 2 was obtained in the same manner as in Example 1 except that it was formed by pressing a mold. In the cryopreservation jig of Example 2, the length between the center of gravity of the image pattern and each vertex was 0.71 mm for the outer square and 0.35 mm for the inner square.

(Example 3)
In the production of the cryopreservation jig of Example 1, the image pattern 4c shown in FIG. 4 (a regular hexagon and a square having the same center of gravity but different lengths of the center of gravity and the apex (both the regular hexagon and the square). A jig for cryopreservation of Example 3 was obtained in the same manner as in Example 1 except that the dotted line))) was formed by pressing the mold. In the cryopreservation jig of Example 3, the length between the center of gravity of the outer regular hexagon and each vertex of the image pattern is 0.5 mm, and the length between the center of gravity of the inner square and each vertex is 0. It was set to 35 mm.

(Example 4)
In the production of the cryopreservation jig of Example 1, the image pattern 4d shown in FIG. 5 (a square having the same center of gravity but different in the radius of the circle and the distance between the center of gravity and the apex (which of the circle and the square). The cryopreservation jig of Example 4 was obtained in the same manner as in Example 1 except that the))) was formed by pressing the mold. In the cryopreservation jig of Example 4, the length between the center of gravity of the outer square of the image pattern and each vertex was 0.71 mm, and the radius of the inner circle was 0.25 mm.

(Example 5)
In the production of the cryopreservation jig of Example 1, the image pattern 4e (two ellipses having the same center of gravity but different minor and major diameters (ellipses formed by dotted lines)) shown in FIG. 6 is pressed by a mold. A jig for cryopreservation of Example 5 was obtained in the same manner as in Example 1 except that it was formed. In the cryopreservation jig of Example 5, the outer ellipse of the image pattern has a major axis of 1.5 mm and a minor axis of 1.0 mm, and the inner ellipse has a major axis of 1.05 mm and a minor axis of 0.75 mm. bottom.

(Comparative Example 1)
In the production of the cryopreservation jig of Example 1, the same procedure as in Example 1 except that the image pattern 4f (single circle (circle formed by the dotted line)) shown in FIG. 7 is formed by pressing the mold. , A jig for cryopreservation of Comparative Example 1 was obtained. In the cryopreservation jig of Comparative Example 1, the diameter of the circle of the image pattern was set to 1 mm.

(Comparative Example 2)
In the production of the cryopreservation jig, the image pattern 4 g shown in FIG. 8 (three circles having the same center of gravity and different diameters (all three circles are formed by solid lines having a line width of 50 μm)) is formed. A jig for cryopreservation of Comparative Example 2 was obtained in the same manner as in Example 1 except that it was formed by pressing a mold. In the cryopreservation jig of Comparative Example 2, the diameter of the circle of the image pattern is 0.75 mm for the outermost circle, 0.5 mm for the second outer circle, and 0.25 mm for the innermost circle. bottom. The pattern has no non-image areas.

For the cryopreservation jigs of Examples 1 to 5 and Comparative Examples 1 and 2, the ease of adjusting the storage liquid amount is determined, and the storage liquid absorbability and the adjustment liquid amount of each of the cryopreservation jigs are a guideline. It was evaluated from the three viewpoints of easy understanding and embryo recovery.

<Evaluation of storage solution absorbency>
At the position of the center of gravity of each image pattern of the preservative liquid absorber in the mounting portion of the cryopreservation jig of Examples 1 to 5 and Comparative Examples 1 to 2, the diameter is located at the position of the center of gravity of each image pattern under a stereomicroscopic observation using a stripper pipette. One 100 μm mouse embryo and 0.2 μl of FUJIFILM Irvine Scientific Vit Kit vitrification solution, which is generally used for vitrification and preservation of cells or tissues as a preservation solution, were placed simultaneously. Observe how the excess preservation solution around the embryo is absorbed by the preservation solution absorber, and use the preservation solution absorber of each cryopreservation jig without manually removing the excess preservation solution around the embryo. Whether or not a sufficient amount was absorbed was evaluated according to the following criteria. These results are shown in the item of "preservative liquid absorbability" in Table 1.

<Evaluation criteria for preservative absorption>
◯: A sufficient amount of the excess preservation solution around the mouse embryo was absorbed by the preservation solution absorber without manual removal.
X: The excess preservation solution around the mouse embryo was not sufficiently absorbed by the preservation solution absorber, and manual removal was required.

<Easy operation for adjusting the amount of storage liquid>
The mouse embryo and the vitrified solution were simultaneously placed on the cryopreservation jigs of Examples 1 to 5 and Comparative Examples 1 and 2 in the same manner as in the evaluation of the storage solution absorbability described above, and the vitrified solution around the mouse embryo was placed. The amount of vitrified solution around cells or tissues is adjusted to an appropriate amount (preservation remaining around mouse embryos) by using the image pattern on the preservation solution absorber as a marker when is absorbed by the preservation solution absorber. Whether or not it was easy to measure the timing when the amount of liquid became appropriate was evaluated according to the following criteria. This result is shown in the item of "Convenience of operation for adjusting the amount of storage liquid" in Table 1.

<Easy evaluation criteria for adjusting the amount of preservative solution>
◯: The amount of the vitrified liquid can be easily adjusted to an appropriate amount by using the image pattern on the preservation liquid absorber as a mark, and the residual amount of the vitrified liquid can be kept constant in 5 trials. did it.
Δ: The amount of the vitrified liquid could be adjusted to an appropriate amount by using the image pattern on the preservation liquid absorber as a mark, but the residual amount of the vitrified liquid could be kept constant at least once during five trials. could not.
X: The amount of the vitrifying liquid could not be adjusted appropriately by using the image pattern on the storage liquid absorber as a mark.

<Recoverability of mouse embryos>
The cryopreservation jigs of Examples 1 to 5 and Comparative Examples 1 and 2 were subjected to the same preservative liquid amount adjustment operation as described above, and immediately after the vitrification liquid amount was adjusted, the cryopreservation jig was cooled with a cooling solvent (liquid). It was put into nitrogen). If the amount of vitrification liquid could not be adjusted to an appropriate amount, it was put into a cooling solvent in that state. Then, the cryopreservation jig was taken out from the cooling solvent (liquid nitrogen), and the mounting portion was immersed in a Vit Kit thaw solution manufactured by FUJIFILM Irvine Scientific Co., Ltd. at a temperature of 37 ° C., and the recoverability of the mouse embryo at the time of thawing was described below. It was evaluated according to the criteria of. These results are shown in the item of "embryo recoverability" in Table 1.

<Evaluation criteria for recoverability of mouse embryos>
◯: Mouse embryos could be recovered in the lysate without any special operation in all 5 of the 5 trials.
Δ: It was necessary to spray the mouse embryo with a pipette to collect the mouse embryo once in five trials.
X: It was necessary to perform an operation of spraying the lysate with a pipette on the mouse embryos in order to collect the mouse embryos at least 2 times out of 5 trials. Or, when the mouse embryo is unintentionally detached at least once out of 5 trials (an event that occurs when the residual amount of preservative solution around the embryo is too large), and the mouse embryo is lost under microscopic observation and cannot be recovered. was there.

From the results in Table 1, according to the present invention, there is a cryopreservation jig that is excellent in preservative liquid absorption and cell or tissue retrievability, and can easily adjust the amount of preservative liquid in the cell or tissue freezing operation. It turns out that it can be obtained.

The cryopreservation jig of the present invention was collected from iPS cells, ES cells, commonly used cultured cells, and living organisms, in addition to embryo transfer, artificial insemination, and artificial insemination of domestic animals such as cattle and animals. It can be used for cryopreservation of cells or tissues for examination or transplantation, cells or tissues cultured in vitro.

1 Grip part 2 Placement part (preservative liquid absorber)
3 Jig for cryopreservation 4 4a-4g Image pattern

Claims (1)

The placement portion on which the cells or tissues are placed has a porous preservation solution absorber, and is composed of a plurality of circles or ellipses having the same center of gravity and different diameters on either the front or back surface of the preservation solution absorber. Image pattern, image pattern composed of multiple polygons with the same center of gravity but different lengths of the center of gravity and vertices, or image pattern composed of a combination of circles or ellipses and polygons with the same center of gravity. A cryopreservation jig having at least one of the above, wherein the image pattern is formed by discontinuous lines.

Images